The present invention relates generally to the regulation of gene expression and more specifically to a method of determining the DNA methylation status of CpG sites in a given locus and correlating the methylation status with the presence of a cell proliferative disorder.
DNA methylases transfer methyl groups from the universal methyl donor S-adenosyl methionine to specific sites on the DNA. Several biological functions have been attributed to the methylated bases in DNA. The most established biological function for methylated DNA is the protection of DNA from digestion by cognate restriction enzymes. The restriction modification phenomenon has, so far, been observed only in bacteria. Mammalian cells, however, possess a different methylase that exclusively methy-lates cytosine residues that are 5xe2x80x2 neighbors of guanine (CpG). This modification of cytosine residues has important regulatory effects on gene expression, especially when involving CpG rich areas, known as CpG islands, located in the promoter regions of many genes.
Methylation has been shown by several lines of evidence to play a role in gene activity, cell differentiation, tumorigenesis, X-chromosome inactivation, genomic imprinting and other major biological processes (Razin, A., H., and Riggs, R. D. eds. in DNA Methylation Biochemis-try and Biological Significance, Springer-Verlag, New York, 1984). In eukaryotic cells, methylation of cytosine residues that are immediately 5xe2x80x2 to a guanosine, occurs predominantly in CG poor regions (Bird, A., Nature, 321:209, 1986). In contrast, CpG islands remain unmethylated in normal cells, except during X-chromosome inactivation (Migeon, et al., supra) and parental specific imprinting (Li, et al., Nature 366:362, 1993) where methylation of 5xe2x80x2 regulatory regions can lead to transcriptional repression. Do novo methylation of the Rb gene has been demonstrated in a small fraction of retinoblastomas (Sakai, et al., Am. J. Hum. Genet., 48:880, 1991), and recently, a more detailed analysis of the VHL gene showed aberrant methylation in a subset of sporadic renal cell carcinomas (Herman, et al., Proc. Natl. Acad. Sci., U.S.A., 91:9700, 1994). Expression of a tumor suppressor gene can also be abolished by de novo DNA methylation of a normally unmethylated CpG island (Issa, et al., Nature Genet., 7:536, 1994; Herman, et al., supra; Merlo et al., Nature Med., 1:686, 1995; Herman, et al., Cancer Res., 56:772, 1996; Graff, et al., Cancer Res., 55:5195, 1995; Herman, et al., Cancer Res., 55:4525, 1995).
Human cancer cells typically contain somatically altered nucleic acid, characterized by mutation, amplification, or deletion of critical genes. In addition, the nucleic acid from human cancer cells often display somatic changes in DNA methylation (E. R. Fearon, et al., Cell, 61:759, 1990; P. A. Jones, et al., Cancer Res., 46:461, 1986; R. Holliday, Science, 238:163, 1987; A. De Bustros, et al., Proc. Natl. Acad. Sci., USA, 85:5693, 1988); P. A. Jones, et al., Adv. Cancer Res., 54:1, 1990; S. B. Baylin, et al., Cancer Cells, 3:383, 1991; M. Makox, et al., Proc. Natl. Acad. Sci., USA, 89:1929, 1922; N. Ohtani-Fujita, et al., Onco-gene, 8:1063, 1993). However, the precise role of abnormal DNA methylation in human tumorigenesis has not been established. Aberrant methylation of normally unmethylated CpG islands has been described as a frequent event in immortized and transformed cells, and has been associated with transcriptional inactivation of defined tumor suppressor genes in human cancers. In the development of colorectal cancers (CRC), a series of tumor suppressor genes (TSG) such as APC, p53, DCC and DPC4 are inactivated by mutations and chromosomal deletions (reviewed in Kinzler and Vogelstein 1996). Some of these alterations result from a chromosomal instability phenotype described in a subset of CRC (Lengauer et al., 1997a). Recently, an additional pathway has been shown to be involved in a familial form of CRC, hereditary non-polyposis colorectal cancer. The cancers from these patients show a characteristic mutator phenotype which causes microsatellite instability (MI), and mutations at other gene loci such as TGG-beta-RII (Markowitz et al., 1995) and BAX (Rampino et al., 1997). This phenotype usually results from mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 (reviewed by Peltomaki, and de la Chapelle, 1997). A subset of sporadic CRC also show MI, but mutations in MMR genes appear to be less frequent in these tumors (Liu et al., 1995; Moslein et al., 1996).
Another molecular defect described in CRC is CpG island (CGI) methylation. CGIs are short sequences rich in the CpG dinucleotide and can be found in the 5xe2x80x2 region of about half of all human genes. Methylation of cytosine within 5xe2x80x2 CGIs is associated with loss of gene expression and has been seen in physiological conditions such as X chromosome inactivation and genomic imprinting (reviewed in Latham, 1996). Aberrant methylation of CGIs has been detected in genetic diseases such as the fragile-X syndrome, in aging cells and in neoplasia. About half of the tumor suppressor genes which have been shown to be mutated in the germline of patients with familial cancer syndromes have also been shown to be aberrantly methylated in some proportion of sporadic cancers, including Rb, VHL, p16, hMLH1, and BRCA1 (reviewed in Baylin, S. B., Herman, J. G., Graff, J. R., Vertino, P. M. and Issa, J. P. Alterations in DNA methylation: a fundamental aspect of neoplasia, Adv. Cancer Res. 72:141-196 1998). TSG methylation in cancer is usually associated with (1) lack of gene transcription and (2) absence of coding region mutation. Thus it has been proposed that CGI methylation serves as an alternative mechanism of gene inactivation in cancer.
The causes and global patterns of CGI methylation in human cancers remain poorly defined. Aging could play a factor in this process because methylation of several CGIs could be detected in an age-related manner in normal colon mucosa as well as in CRC (Issa, J. P., Vertino, P. M., Boehm, C. D., Newsham, I. F. and Baylin, S. B. Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet. 7:536-540, 1994). In addition, aberrant methylation of CGIs has been associated with the MI phenotype in CRC as well as specific carcinogen exposures (Issa et al., 1996) supra. However, an understanding of aberrant methylation in CRC has been somewhat limited by the small number of CGIs analyzed to date. In fact, previous studies have suggested that large numbers of CGIs are methylated in immortalized cell lines (Antequera, F., Boyes, J. and Bird, A. High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 62:503-514, 1990), and it is not well understood whether this global aberrant methylation is caused by the cell culture conditions or whether they are an integral part of the pathogenesis of cancer.
Most of the methods developed to date for detection of methylated cytosine depend upon cleavage of the phosphodiester bond alongside cytosine residues, using either methylation-sensitive restriction enzymes or reactive chemicals such as hydrazine which differentiate between cytosine and its 5-methyl derivative. Genomic sequencing protocols which identify a 5-MeC residue in genomic DNA as a site that is not cleaved by any of the Maxam Gilbert sequencing reactions have also be used, but still suffer disadvantages such as the requirement for large amount of genomic DNA and the difficulty in detecting a gap in a sequencing ladder which may contain bands of varying intensity.
Mapping of methylated regions in DNA has relied primarily on Southern hybridization approaches, based on the inability of methylation-sensitive restriction enzymes to cleave sequences which contain one or more methylated CpG sites. This method provides an assessment of the overall methylation status of CpG islands, including some quantitative analysis, but is relatively insensitive and requires large amounts of high molecular weight DNA.
Another method utilizes bisulfate treatment of DNA to convert all unmethylated cytosines to uracil. The altered DNA is amplified and sequenced to show the methylation status of all CpG sites. However, this method is technically difficult, labor intensive and without cloning amplified products, it is less sensitive than Southern analysis, requiring approximately 10% of the alleles to be methylated for detection.
Identification of the earliest genetic changes in tumorigenesis is a major focus in molecular cancer research. Diagnotic approaches based on identification of these changes are likely to allow implementation of early detection strategies and novel therapeutic approaches targeting these early changes might lead to more effective cancer treatment.
The present invention is based on the finding that several genes are newly identified as being differently methylated in cancer. This seminal discovery is useful for cancer screening, risk-assessment, prognosis, minimal-residual disease identification, staging and identification of therapeutic targets. The identification of new genes that are methylated in cancer, aging or diseases associated with aging increases the likelihood of finding genes methylated in a particular cancer; increases the sensitivity and specificity of methylation detection; allows methylation profiling using multiple genes; and allows identification of new targets for therapeutic intervention. The invention also provides a newly identified gene that is a target for hypermethylation in human tumors. This new gene, as well as genes newly identified as hypermethylated in cancer and aging or aging diseases provides markers which can be used diagnostically, prognostically and therapeutically over the course of such disorders.
In a first embodiment, the invention provides a nucleic acid molecule comprising a coding region for a T-type calcium channel, CACNA1G, and regulatory sequences associated therewith. The discovery of CpG islands, and in particular, methylated CpG islands in the region approximately 300-800 base pairs upstream for the CACNA1G translation initiation start site, led to a method of the present invention for correlating methylated CpG islands with various cancers. In one aspect of this embodiment, the nucleic acid molecule encoding CACNA1G and the associated regulatory sequences and CpG-rich regions include the nucleic acid sequence set forth in SEQ ID NO:51 (FIG. 3A). Also provided is a polypeptide having an amino acid sequence as set forth in SEQ ID NO:52 and FIG. 3B. The methylation state of CpG islands in CACNA1G, associated regulatory regions, and other genes is indicative of the presence of a cellular proliferative disorder in a subject from which the CpG-containing nucleic acid is isolated.
In another embodiment, there are provided methods for detecting a cellular proliferative disorder in a subject. The subject may have or be at risk of having a cellular proliferative disorder. The method of the invention is useful for diagnostic as well as prognostic analyses. One method for detecting a cellular proliferative disorder in a subject includes contacting a nucleic acid-containing specimen from the subject with an agent that provides a determination of the methylation state of at least one gene or associated regulatory region of the gene; and identify aberrant methylation of regions of the gene or regulatory region, wherein aberrant methylation is identified as being different when compared to the same regions of the gene or associated regulator region in a subject not having the cellular proliferative, thereby detecting a cellular proliferative disorder in the subject. The method includes multiplexing by utilizing a combination of primers for more than one loci, thereby providing a methylation xe2x80x9cprofilexe2x80x9d for more than one gene or regulatory region.
For the first time, the invention provides methylated forms of the following genes and/or their associated regulatory sequences: APOB, CACNA1G, CDX2, EGFR, FBN1, GPR37, HSPA6, IQGAP2, KL, PAR2, PITX2, PTCH, SDC1, and SDC4 (see Table 5). In addition, the invention provides the CpG-rich regions from these genes that are hypermethylated (see FIGS. 4A-4F (SEQ ID NO:105-119).
Invention methods include determining, in a nucleic acid-containing specimen taken from a subject, the methylation state of a gene or regulatory sequences associated therewith, wherein the expression or non-expression of the gene is associated with the presence of the cellular proliferative disorder, and identifying as having a cellular proliferative disorder a subject that has aberrant methylation of regions of the gene or associated regulatory sequences when compared to the same regions of the gene in a subject not having the cellular proliferative disorder. In one aspect of this embodiment, the methylated regions of the gene and associated regulatory sequences are contained within CpG islands (i.e., CpG rich regions). In particular, the aberrant methylation typically includes hypermethylation as compared with the same regions of the gene or regulatory sequences in a subject not having the cellular proliferative disorder.
Determining the methylation state of the gene includes contacting the nucleic acid-containing specimen with an agent that modifies unmethylated cytosine, amplifying a CpG-containing nucleic acid in the specimen by means of CpG-specific oligonucleotide primers, wherein the oligonucleotide primers distinguish between modified methylated and nonmethylated nucleic acid, and detecting the methylated acid based on the presence or absence of amplification products produced in said amplifying step. The method includes optionally contacting the amplification products with a methylation sensitive restriction endonuclease. Other methods for determining methylation status of a gene and/or regulatory sequences are well known in the art and are described more fully herein.
In another embodiment, the present invention provides a method of treating a cell proliferative disorder associated with CACNA1G or other methylated genes described herein, comprising administering to a subject with the disorder, a therapeutically effective amount of reagent which modulates CACNA1G or other methylated genes"" expression. For example, since CACNA1G -associated disorders typically involve hypermethylation of CACNA1G polynucleotide sequence, a polynucleotide sequence which contains a non-methylatable nucleotide analog is utilized for treatment of a subject. Further, the invention provides a method of gene therapy comprising introducing into cells of a host subject, an expression vector comprising a nucleotide sequence encoding CACNA1G or other methylated genes described herein, in operable linkage with a promoter.
In another embodiment of the present invention there is provided a kit useful for the detection of a cellular proliferative disorder in a subject having at risk for having a cellular proliferative disorder. Invention kits include a carrier means compartmentalized to receive a sample, one or more containers comprising a first container containing a reagent which modifies unmethylated cytosine and a second container containing primers for amplification of a CpG-containing nucleic acid, wherein the primers distinguish between modified methylated and nonmethylated nucleic acid, and optionally, a third container containing a methylation sensitive restriction endonuclease.